2.3 | Miniaturization and intracellular accumulation of
droplets
Next, the lipid-coated PFH droplets were miniaturized and examined for
uptake into living cells through endocytosis. The diameter of endosomes
has been reported to be less than 120 nm[5], and
we wanted the droplets to be smaller than this. Therefore, the droplets
were extruded through 30 nm pore filters, and their average diameter was
determined to be 66.8 nm using dynamic light scattering (Figure 2B). The
present PCNDs were modified with biotinylated and fluorescently labeled
9E5 through cross-linking with NeutrAvidin. Alexa Fluor 647 (AF647) was
used for labeling 9E5. In addition, a fluorescently labeled PEG-lipid
(tetramethyl rhodamine (TAMRA)-labeled PEG-lipid, DPPE-PEG-TAMRA)
(Figure 1; also see Supporting Information, Figure S1) was incorporated
into the lipid coating of the droplets to visualize the PCNDs. In this
study, human colon carcinoma DLD1 cells were used as the model
EREG-expressing cancer cells. After the treatment of DLD1 cells with
9E5-conjugated PCNDs for 2 h at 37 ᵒC, the fluorescence of both TAMRA
and AF647 was observed using confocal fluorescence microscopy at almost
the same location inside the cells (Figure 2C). In the case of
non-conjugated PCNDs, no fluorescence was observed inside the cells
(Figure 2D). These results indicate that the present PCNDs were taken up
into living cells due to 9E5 conjugation, as previously
reported[30,31].